Cloning, expression and use of acid phospholipases

ABSTRACT

The invention relates to a DNA sequence, which codes for a polypeptide having phospholipase activity essentially without lipase activity, characterized in that the DNA sequence is selected from a) DNA sequences that comprise a nucleotide sequence according to SEQ ID NO: 1, b) DNA sequences that comprise the coding sequence according to SEQ ID NO: 1, c) DNA sequences that code for the protein sequence according to SEQ ID NO: 2, d) DNA sequences that are coded for by the plasmid pPL3940-Topo2.5 with the restriction map according to FIG.  7 , which is deposited under accession number DSM 22741, e) DNA sequences that hybridize under stringent conditions with one of the DNA sequences according to a), b), c) or d), f) DNA sequences that are related to the DNA sequences according to a), b), c), d) or e) due to the degeneration of the genetic code, and g) complementary strands to the sequences according to a) to f), wherein the DNA sequence is preferably derived from  Aspergillus , and more preferably from  Aspergillus fumigatus , and a polypeptide having phospholipase activity essentially without lipase activity selected from a) a polypeptide which is coded for by the coding part of a DNA sequence as defined above, b) a polypeptide having the sequence according to SEQ ID NO: 2 or a sequence derived therefrom, which may be obtained by substitution, addition, deletion of one or more amino acid(s), c) a polypeptide having a sequence that has at least 83% identity with the amino acids 1 to 299 of SEQ ID NO: 2, d) a polypeptide which is coded for by a nucleic acid sequence which hybridizes under stringent conditions with (i) nucleotides 55 to 1106 of SEQ ID NO: 1, (ii) the cDNA sequence contained in nucleotides 55 to 1106 of SEQ ID NO: 1, (iii) a partial sequence of (i) or (ii) composed of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii) or (iii), e) a variant of the polypeptide having SEQ ID NO: 2, comprising a substitution, deletion and/or insertion of one or more amino acid(s), f) allelic variants to amino acid sequences a) to e).

RELATED APPLICATIONS

This application is a divisional of, and claims priority under 35 U.S.C.§120 to, U.S. patent application Ser. No. 13/502,945 filed Apr. 19,2012, which is a national stage application, filed under 35 U.S.C. §371,of International Application No. PCT/EP2010/066234 filed Oct. 27, 2010,which claims the benefit under 35 U.S.C. §119 to German PatentApplication No. 102009051013.3, filed Oct. 28, 2009, the entire contentsof each of which are hereby incorporated herein by reference in theirentirety.

The invention relates to new DNA sequences which code for polypeptideshaving phospholipase activity essentially without lipase activity. Theinvention further relates to new polypeptides having phospholipaseactivity essentially without lipase activity. These polypeptides areacid phospholipases with low molecular weight, high thermostability andhigh temperature resistance. Furthermore, these polypeptides are activewithin a broad pH range. Moreover, the invention also relates to the useof these phospholipases for reducing phosphorus-containing compounds,for example in the production of edible oils, as well as to the use ofthese phospholipases as bakery improver, animal feed additive, additivein the processing of textile raw materials, etc.

Crude vegetable oils contain associated material (such as free fattyacids, phospholipids, heavy metals, colorants . . . ) which affect thequality and shelf life of the oil and complicate its further processingdue to the hydrolytic and oxidative modification of lipids duringstorage. Therefore, it is necessary to refine the crude vegetable oilsafter their extraction in order to remove the impurities. In theproduction of high-quality edible oils the refining process comprisesthe steps of degumming, bleaching and deodorization.

The first step in the process of refining oils is degumming. During thedegumming process gummy substances, primarily phospholipids, are removedwhich negatively affect the taste of the oil and which disturb thefurther steps of the oil refining process.

Since the phosphorous content is an indicator for the degree ofdegumming the quality of the degummed oil obtained may be assessed bydetermining the content of the remaining phosphorus. For the furthersteps of the refining process degummed oil with a content of theremaining phosphorus of less than 10 ppm is required.

Phospholipids are complex, phosphorus-containing lipids. Phospholipids,such as phosphatidyl choline or lecithin, consist of a glycerinestructure which is esterfied with fatty acids at positions sn-1 and sn-2and with an ester-bound phosphate group at position sn-3. The phosphategroup itself may be esterfied with e.g. a primary alcohol group. Atpositions sn-1 and sn-2 natural phospholipids contain different fattyacid chains which, in the case of plants, are primarily polyunsaturatedacyl chains. There are two kinds of phospholipids: hydratable ones andnon-hydratable ones. In order to remove the phospholipids differentmethods are used.

The simplest method is water-degumming. During this process hydratablephospholipids may be washed out with the help of water and are thusremoved from the oil. Depending on the type and quality of the crude oilthe degummed oil still contains 80 to 200 ppm of phosphorus after thisprocess.

In the acid degumming process the oil is treated with acid. The acidtransforms non-hydratable phospholipids into hydratable phospholipids.The hydratable phospholipids become oil-insoluble. An oil-insolubleslurry is formed which is removed from the oil by means ofcentrifugation or filtration. After this process the content of theremaining phosphorus in the oil is approximately 25 to 100 ppm.

Enzymatic degumming offers an efficient, cost-effective andenvironmentally friendly process for gently removing phospholipids fromedible oil.

Phospholipases are enzymes which cleave phospholipids and are dividedaccording to their enzymatic cleavage sites at the phospholipid intoacylhydrolases (phospholipase A1, A2 and B) and phosphodiesterases(phospholipase C and D). The phospholipase A1 (EC 3.1.1.32) hydrolyzesthe fatty acid at the sn-1 position of phospholipid molecules and thephospholipase A2 (EC 3.1.1.4) specifically cleaves the sn-2 ester bondfrom the phospholipid. As reaction products the lysophospholipid and thefree fatty acid are formed. Phospholipase B (EC 3.1.1.5) unspecificallycleaves the fatty acid at both sn-1 and sn-2 positions. Phospholipase C(EC 3.1.4.10) hydrolyzes the phosphate ester bond between glycerine andthe phosphate group to form phosphate monoester and diacylglycerol.Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of the terminalphosphodiester bond to form the cleavage products phosphatidic acid andcholine.

The extraction of phospholipase A2 from bovine and porcine pancreas andfrom the toxin of honeybees or several snake species is already known.Phospholipase may also be extracted from microorganisms such as bacteriaand fungi and may be produced with a sufficient yield by means ofrecombinant techniques.

In patent EP 0 513 709 an efficient enzymatic degumming process ispresented for the first time. The new process uses the enzymephospholipase A2 for the first time which cleaves the fatty acid atposition sn-2 of phospholipids. The process was tested with soybean andrapeseed oil having a phosphorus content of 72 to 110 ppm. The reactionbatch contained up to 5 wt. % (w/w) water in relation to the oil and wasincubated at a pH value of 5.0 to 5.5 at 40° C. or alternatively 60° C.for up to 5 hours. The lysophospholipid produced may be removed from theoil by means of centrifugation. After the process the content of theremaining phosphorus in the degummed oil is less than 5 ppm.

During the degumming process the oil is mixed with a defined amount ofwater. Then, the enzyme-containing aqueous phase and the oily phase aremixed in order to allow the enzyme to act. The amount of water shouldhereby be as small as possible. The use of large amounts of water leadsto increased energy consumption and increased disposal costs. Thereforethe process of enzymatic degumming with a low water content (2%) isalready advantageous in this regard.

In US2007/134777 it is stated that the enzymatic degumming of vegetableoil with the help of phospholipase A1 is carried out at a pH value ofbetween 4.0 and 5.0. The optimal pH value for carrying out this processis between 4.5 and 5.0. In this pH range the released calcium and/ormagnesium ions may combine with other chemicals (anions) of the reactionbuffer to form hardly soluble salts which deposit on the surface of thereactors and thus soil the device. The removal of such soiling and thecleaning of the device is laborious. In order to reduce the soiling ofthe interior of the device the reaction is preferably carried out at apH value of approximately 4. However the enzyme becomes less active orfunctionally inactive if the pH value of the reaction is loweredfurther.

EP 0 904 357 describes that in Aspergillus niger a phospholipase A wasfound for the degumming of edible oil.

WO2008/040466 describes that a phospholipase having a molecular weightof 65 kDa may be used in Aspergillus fumigatus for the degumming ofedible oil with a water content of 5% at temperatures of up to 65° C.

EP 1 788 080 describes the use of phospholipase C of Bacillus cereus forthe degumming of oil in 6 hours at 60° C. with a water content of 15% inrelation to the oil. After the process the content of the remainingphosphorus is less than 5 ppm.

In WO2008/094847 it is described that the reaction time of the oildegumming process may be reduced to 30 min under the reaction of thephospholipase A1 or A2 with the phospholipase C, respectively.

Phospholipases are also manifoldly used in the food industry and theanimal feed industry, e.g., for the preparation of dough, for thepreparation of bakery products, for increasing the yield of cheeseproduction etc. Thus, there is a need for phospholipases which may beversatilely used in technology.

In biotechnology phospholipases are used as biocatalysts for theextraction of phospholipids. Phospholipids are polar lipids and act asemulgators due to their lipophilic and hydrophilic structural features.Examples are the use of phospholipases in the production of modifiedlecithins, as food emulsions in the production of sauces, mayonnaise andsalad dressing, in the production of instant powders, such as milk,cocoa and coffee powders, as flow improvers in the production ofchocolate and as food supplements. In pharmaceutical and cosmeticindustry phospholipids and lysophospholipids are used for the productionof cremes, lotions, gels and liposome formulations.

Lecithin is also required for the production of varnishes, paints,magnetic tapes, speciality papers, leather and textiles. Phospholipasesare also used in textile industry for “bioscouring” to clean the plantfibre before further process steps such as colorization are carried out.A mixture of phospholipase together with other enzymes may also be usedhere. The other enzymes may be selected from the group of cellulases,hemicellulases, pectinases, proteases and oxidoreductases.

Thus there is a constant need for phospholipases having a possibly largeand optimized field of application in the art.

The present invention is therefore based on the object of providingproteins or polypeptides with improved phospholipase properties. The newphospholipases are particularly not to show lipase activity relevant intechnological processes. In particular, the proteins havingphospholipase activity are to be active over a large pH range and are tobe highly temperature-resistant.

Moreover, the production of the proteins having phospholipase activityis to be simple, cost-efficient and commercial. Furthermore, expressionconstructs according to the invention which are suitable for theproduction of the proteins having phospholipase activity are to beprovided.

The aforementioned objects are solved by means of a DNA sequence, whichcodes for a polypeptide having phospholipase activity essentiallywithout lipase activity, characterized in that the DNA sequence isselected from a) DNA sequences that comprise a nucleotide sequenceaccording to SEQ ID NO: 1, b) DNA sequences that comprise the codingsequence according to SEQ ID NO: 1, c) DNA sequences that code for theprotein sequence according to SEQ ID NO: 2, d) DNA sequences that arecoded for by the plasmid pPL3949-Topo2.5 with the restriction mapaccording to FIG. 7, which is deposited under accession number DSM22741, e) DNA sequences that hybridize under stringent conditions withone of the DNA sequences according to a), b), c) or d), f) DNA sequencesthat are related to the DNA sequences according to a), b), c), d) or e)due to the degeneration of the genetic code, and g) complementarystrands to the sequences according to a) to f).

The invention further relates to a polypeptide having phospholipaseactivity essentially without lipase activity selected from a) apolypeptide which is coded for by the coding part of a DNA sequence asdefined above, b) a polypeptide having the sequence according to SEQ IDNO: 2 or a sequence derived therefrom, which may be obtained bysubstitution, addition, deletion of one or more amino acids, c) apolypeptide having a sequence that has at least 83% identity with theamino acids 1 to 299 of SEQ ID NO: 2, d) a polypeptide which is codedfor by a nucleic acid sequence which hybridizes under stringentconditions with (i) nucleotides 55 to 1106 of SEQ ID NO: 1, (ii) thecDNA sequence contained in nucleotides 55 to 1106 of SEQ ID NO: 1, (iii)a partial sequence of (i) or (ii) composed of at least 100 nucleotides,or (iv) a complementary strand of (i), (ii) or (iii), e) a variant ofthe polypeptide having SEQ ID NO: 2, comprising a substitution, deletionand/or insertion of one or more amino acids, f) allelic variants toamino acid sequences a) to e).

Furthermore, the invention relates to expression constructs or hoststhat are able to express polypeptides having phospholipase activityaccording to the invention. Moreover, the invention also relates to therespective expression plasmids and vectors. Furthermore, the inventionrelates to processes for the degumming of vegetable oil using thepolypeptides according to the invention as well as to the use of thepolypeptides according to the invention in the field of food technology,in particular for the preparation of dough, bakery products or dairyproducts or in animal nutrition and in the processing of textile rawmaterials, the so-called scouring or bioscouring.

According to a further embodiment the invention relates to a polypeptidehaving phospholipase activity essentially without lipase activitycharacterized in that it has a molecular weight in the range of 28 to 30kDA and preferably of approximately 28.6 kDa, a broad pH optimum and ahigh temperature resistance and that it may be isolated from an organismof the genus Aspergillus.

High temperature resistance in this context means that after 6 hours ata temperature of 60° C. under the conditions of the oil degummingprocess with a low water content of 2% the enzyme retains its activityto an industrially exploitable extent. The enzyme retains its activityto an industrially exploitable extent if it produces oils of which thecontents of the remaining phosphorus are technologically insignificant,so to speak. Preferably the content of the remaining phosphorus in theenzymatically degummed oil is less than 10 ppm, more preferably lessthan 5 ppm.

Surprisingly it was found that a DNA sequence which codes for apolypeptide having phospholipase activity essentially without lipaseactivity which has a low molecular weight as well as a high temperatureresistance may be isolated from a strain of the genus Aspergillusfumigatus. This phospholipase is an acid phospholipase derived from afilamentous fungus with a calculated molecular weight of approximately28.6 kDa, which is able to hydrolyze at least one of the two fatty acidsfrom lecithin.

In contrast to the polypeptides having phospholipase activity known fromthe state of the art, the phospholipases according to the invention havea high temperature resistance (at 60° C.) and can, thus, also bebeneficially used in processes of enzymatic degumming with a low watercontent of 2% (in relation to oil) and at a pH value of 4.0. This is ofparticular economic interest since the temperature of the oil does notfirst have to be lowered in the degumming processes to make enzymaticdegumming without inactivation of the enzyme possible, and subsequentlythe temperature of the oil has again to be increased in order todecrease the viscosity of the oil for the centrifugation step used forseparating the oily phases from the aqueous phases. The temperatureresistance of the polypeptides having phospholipase activity accordingto the invention is also advantageous for other applications in thefield of food technology and animal nutrition and in textile processing,respectively.

Phospholipases known from the state of the art are excluded from thescope of the invention.

Therefore it is particularly advantageous that the phospholipasesderived from A. fumigatus according to the invention are active within alarge pH range of 3 to 5 or show a broad activity optimum within thisrange. Therefore the phospholipases according to the invention do notonly have the advantage that they essentially have no lipase activity.They additionally develop their enzyme activity over a large pH rangeand may thus be used over a large pH range.

Enzymes having phospholipase activity (phospholipase A, B, C or D) fromAspergillus fumigatus have so far been mentioned in several publications(Birch et al., Comparison of extracellular phospholipase activities inclinical and environmental Aspergillus fumigatus isolates, 2004, MedMycol 42(1): 81-86; Rementeria et al., Genes and molecules involved inAspergillus fumigatus virulence, 2005, Rev Iberoam Micol 22(1): 1-23)but have not been exactly characterized. From the genome sequence ofAspergillus fumigatus (Nierman et al., Genomic sequence of thepathogenic and allergenic filamentous fungus Aspergillus fumigatus,2005, Nature, 438(7071): 1151-6) several hypothetical phospholipase (A,B, C, D) and lysophospholipase genes were derived under the term“conceptual translation”. Thus, e.g. one hypothetical phospholipase Ahaving 241 amino acids and three lysophospholipases Plb1, Plb2 and Plb3having a high molecular weight were displayed. Furthermore, the databaseshows a small hypothetical extracellular lipase having 299 amino acidsand a calculated molecular weight of approximately 28.5 kDa (GenBankEAL86100) as well as several lipases having between 409 and 587 aminoacids.

Shen et al. (Characterisation and expression of phospholipase B from theopportunistic fungus Aspergillus fumigatus, 2004, FEMS Microbol Lett239(1): 87-93) succeeded in cloning and characterizing 3 phospholipase Bgenes from Aspergillus fumigatus. The secreted proteins AfPL1 having 633amino acids and AfPL3 having 630 amino acids have a molecular weight ofapproximately 68 kDa. The protein AfPL2 is a cytosolic protein having588 amino acids and has a molecular weight of approximately 63 kDa.

Moreover, a phospholipase having 633 amino acids (WO2008/040466) and alysophospholipase having 611 amino acids (WO2008/040465) were isolatedfrom Aspergillus fumigatus RH3949.

Furthermore, an acid phospholipase (pI 4.1) having a small molecularweight within the range of 28 to 30 kDa was found in the genome ofAspergillus fumigatus RH3949.

In the protein sequencing process of this “small” phospholipase thefirst 16 amino acids showed 93% identity at the N-terminus with theN-terminal sequence of the hypothetical extracellular lipase having 299amino acids (GenBank EAL86100) from Aspergillus fumigatus Af293. Theidentity of the mature (AA 30-299) sequence of the “small phospholipase”from RH3949 with the sequence of the hypothetical extracellular lipase,however, is only approximately 82%. This was particularly surprisingsince at a comparable site in the genome of a further Aspergillusfumigatus strain (A1163) a sequence having a significantly higheridentity with the hypothetical lipase from Af293 had been found (GenBankEDP1054) (Fedorova et al., Genomic Islands in the Pathogenic FilamentousFungus Aspergillus fumigatus, 2008, PloS Genet. 4. e1000046).

In contrast thereto there is no analogy between two N-terminalphospholipase sequences from Aspergillus fumigatus Af293 (phospholipaseA having 241 amino acids, GenBank EAL85761) and Aspergillus fumigatusRH3949 (phospholipase having pI 4.1).

Therefore the phospholipase according to the invention differs fromknown phospholipases from Aspergillus fumigatus as well as fromsequences annotated as lipases of closely related Aspergillus fumigatusstrains such as Af293.

According to the invention several oligonucleotide primers were derivedand synthesized from the DNA sequence data (GenBank AAHF01000011) forthe hypothetical extracellular lipase (GenBank EAL86100).

Surprisingly it was found that the DNA sequence according to theinvention could be isolated from the Aspergillus strain RH3949 by meansof primers derived from the genome sequence of the strain Af293 (cf.example 4). This was not expected since the strain RH3949 (anenvironmental isolate) used for amplification differs from Af293(clinical isolate) phenotypically and therefore most probably alsogenotypically (sequence). Thus, it was not possible either to amplifythe phospholipase gene from RH3949 by means of other primer pairs havingbonding sites adjacent to N2-3948 and ApaI-3949 on the genomic DNA ofthe strain Af293.

The amplification of the gene was carried out with the help of thepolymerase chain reaction (PCR) from genomic DNA of Aspergillusfumigatus RH3949.

The temperature resistance and the broad pH optimum of the phospholipaseaccording to the invention were surprising and not to be expected on thebasis of the phospholipases described in the state of the art. For noneof the naturally occurring phospholipases derived from filamentous fungias described in the state of the art have these properties beendescribed or merely suggested.

The phospholipase sequence according to SEQ ID NO: 2 according to theinvention was compared to phospholipase sequences of the state of theart. A partial analogy of the amino acid sequence was found with knownamino acid sequences from other Aspergillus strains, i.e. an analogy of60% with a lipase of Aspergillus tubingensis (WO98/45453) or with alysophospholipase from Aspergillus foetidus (EP0808903), 59% with anAspergillus niger phospholipase (WO03/097825, WO98/31790).

The sequence SEQ ID NO: 2 exhibits the highest identity of 82% with ahypothetical sequence for extracellular lipase (GenBank EAL86100) fromAspergillus fumigatus Af293 (Nierman et al., Genomic sequence of thepathogenic and allergenic filamentous fungus Aspergillus fumigatus,2005, Nature, 438(7071): 1151-6).

Surprisingly, under the conditions of the enzymatic degumming of edibleoil the phospholipase isolated from Aspergillus fumigatus RH3949according to the invention does not show any lipase activity relevantfor this process. Furthermore, this enzyme has a remarkably broad pHoptimum and a high temperature resistance in contrast to phospholipasesfrom other Aspergillus strains known so far. Thus, the enzyme accordingto the invention may be used advantageously in a process of enzymaticdegumming of edible oils since it does not hydrolyze any or merelyinsignificant portions of triglyceridebonds in the oil.

Moreover, the invention also relates to polypeptides havingphospholipase activity essentially without lipase activity with asequence which has an identity of at least 83% with the sequenceaccording to SEQ ID NO: 2. Preferably the invention relates to apolypeptide having phospholipase activity with a sequence which has anidentity of at least 83% with the amino acids 1 to 299 of SEQ ID NO: 2.Preferably the degree of identity with the amino acids 1 to 299 of SEQID NO: 2 is at least 90%, more preferably at least 95%, even morepreferably at least 97% and particularly preferably at least 98%provided that the respective sequences have phospholipase activityessentially without lipase activity.

The polypeptides having phospholipase activity according to theinvention do not have any significant lipase activity or are ratheressentially without lipase activity. The polypeptides according to theinvention essentially do not have any lipase activity disadvantageousfor industrial processes of oil degumming, i.e. the polypeptidesaccording to the invention essentially do not show any activity againstlipolytically cleavable compounds in the oil to be degummed. This meansthat under the conditions of the enzymatic degumming of edible oil thephospholipases according to the invention do not have any lipaseactivity relevant for this process. Technologically speaking this meansthat polypeptides having phospholipase activity according to theinvention hydrolyze p-nitrophenyl palmitate as lipase substrate only toan insignificant and/or undetectable extent. For the polypeptides havingphospholipase activity according to the invention the ratio ofphospholipase activity to lipase activity is preferably >1000:1, morepreferably 5000:1 to 10000:1, even more preferably 7000:1 and mostpreferably 7500:1.

The degree of sequence identity is preferably determined in such a waythat the number of residues of the shorter sequence which is involved inthe comparison and has a “corresponding” counterpart in the othersequence is determined. For the purposes of the present invention theidentity is preferably determined in the usual manner by using the usualalgorithms. According to the invention only the cDNAs or amino acids ofthe respective mature proteins are used for the comparison. Similar,preferably identical sequence counterparts were determined according tothe invention as homologue sequences by means of known computerprograms. An example of such a program is the program Clone ManagerSuite, which includes the program part Align Plus and is distributed byScientific & Educational Software, Durham, N.C., U.S.A. A comparison oftwo DNA sequences or amino acid sequences as defined above is therebycarried out under the option local alignment either according to theFastScan-MaxScore method or according to the Needleman-Wunsch method,keeping the default values. The program version “Clone Manager 7 AlignPlus 5” with the functions “Compare Two Sequences/Local Fast Scan-MaxScore/Compare DNA sequences” or for amino acids “Compare TwoSequences/Global/Compare sequences as Amino Acids” was particularly usedto calculate the identity according to the invention. The algorithmsmade available from the following sources were thereby used: Hirschberg,D. S. 1975. A linear space algorithm for computing maximal commonsubsequences. Commun Assoc Comput Mach 18:341-343; Myers, E. W. and W.Miller. 1988. Optimal alignments in linear space. CABIOS 4:1, 11-17;Chao, K-M, W. R. Pearson and W. Miller. 1992. Aligning two sequenceswithin a specified diagonal band. CABIOS 8:5, 481-487.

The invention further relates to addition molecules and/or deletionmolecules of the aforementioned polypeptides having phospholipaseactivity. Thus, a polypeptide having phospholipase activity modifiedaccording to the invention may be elongated by adding further sequencesat the N-terminal and/or C-terminal end, whereby the thus obtained aminoacid sequences still have to show phospholipase activity essentiallywithout lipase activity. Thereby hybrid molecules may be produced whichhave further advantageous properties. For example, suspension proteinsor their native precursor forms may be added to largely secretedproteins, which further enhances secretion efficiency. Moreover, activesequence segments of other enzymes may be added to produce enzymes withmultiple specificity. Furthermore, polar and non-polar sequences may beadded to specifically influence the solubility properties or themembrane mobility of the thus obtained enzyme.

Sequence segments of the polypeptide having phospholipase activity mayalso be deleted according to the invention, keeping the phospholipaseactivity essentially without lipase activity. The mutations, elongationsand shortenings may be carried out in a way known per se and with thehelp of methods well known in the state of the art. Shortenedpolypeptides are often characterized by an enhanced secretion heightcompared to the full-length polypeptides. They may also show higherthermostabilities compared to the full-length polypeptide since theyonly contain the “compressed core”.

The production of such variants is generally known in the state of theart. For example, amino acid sequence variants of the polypeptides maybe produced by mutation in the DNA. Processes for mutagenesis andchanges in the nucleotide sequence are well known in the state of theart (cf. for example Tomic et al. NAR, 18:1656 (1990), Giebel and SprtizNAR, 18:4947 (1990)).

Details on appropriate amino acid substitutions which do not negativelyinfluence the biological activity of the protein of interest can befound in the model by Dayhoff et al., Atlas of Protein Sequence andStructure, Natl. Biomed. Res. Found., Washington, D.C. (1978).Conservative substitutions such as the substitution of an amino acid byanother one with similar properties are preferred. These substitutionsmay be divided into two main groups with altogether four subgroups, anda substitution in each subgroup is referred to as conservativesubstitution, which does preferably not influence the activity or thefolding of the protein.

aliphatic non-polar G A P I L V polar and uncharged C S T M N Q polarand charged D E K R aromatic H F W Y

The expressions “protein”, “peptide” and “polypeptide” are essentiallyused interchangeably. A polypeptide or enzyme having phospholipaseactivity or a phospholipase is to refer to an enzyme which catalyzes therelease of fatty acids from phospholipids, for example, lecithins. Thephospholipase activity may be determined by means of the use of anymeasuring method known per se in which one of these substrates is used.

In connection with the polypeptides according to the invention theexpressions “phospholipase” or phospholipase A are to refer to enzymeshaving phospholipase A1 activity as well as phospholipase A2 activity.Phospholipase A1 or A2 is thereby defined according to the standardenzyme EC classification as EC 3.1.1.32 or 3.1.1.4, respectively.

Phospholipase B or lysophospholipase are polypeptides according to thestandard enzyme EC classification EC 3.1.1.5.

The invention also relates to DNA sequences which code for a polypeptidehaving phospholipase activity, comprising mutations, modifications orvariations of the sequence according to SEQ ID NO: 1. Furthermore, theinvention also relates to sequences which hybridize with theaforementioned sequences under relaxed or stringent conditions. Thefollowing conditions are considered as stringent: hybridization at 65°C., 18 h in dextran sulphate solution (Genescreen-Plus, DuPont),subsequent washing of the filter for 30 min, respectively, at first with6×SSC, twice 2×SSC, twice 2×SSC, 0.1% SDS and finally with 0.2×SSC at65° C. (membrane transfer and detection methods, Amersham).

Furthermore, the invention also relates to DNA sequences which arerelated to the aforementioned sequences according to the invention dueto the degeneration of the genetic code as well as allelic variantsthereof. The degeneration of the genetic code may thereby result fromthe natural degeneration or an especially selected codon usage.Naturally occurring allelic variants may be identified by means ofwell-known techniques of molecular biology such as the polymerase chainreaction (PCR) and hybridization techniques.

The invention also relates to a process for the production of apolypeptide having phospholipase activity using recombinant techniquescomprising the growing of recombinant prokaryotic and/or eukaryotic hostcells which comprise a DNA sequence according to the invention underconditions which promote the expression of the enzyme as well as thesubsequent extraction of the enzyme. The invention further relates tothe use of the polynucleotide sequences according to the invention forthe production of probes for the detection of similar sequences whichcode for corresponding enzymes in other organisms as well as for thetransformation of host cells.

A DNA sequence which codes for a polypeptide according to the inventionmay be used to transform any host cells such as cells of fungi, yeasts,bacteria, plants or mammals. Cells transformed in such a way arecharacterized by a secretion of the phospholipase according to theinvention. The thus produced phospholipase enzyme effects an efficienthydrolysis of the fatty acids from phospholipids.

The invention also relates to expression cassettes which may be used forintroducing the DNA sequence coding for a phospholipase according to theinvention or an open reading frame into a host cell. They preferablycomprise a transcription start region which is connected with the openreading frame. Such an expression cassette may comprise a variety ofrestriction cleavage sites for inserting the open reading frame and/orother DNAs, e.g., a transcription regulator region and/or selectablemarker genes. The transcription cassette comprises in 5→3′ direction ofthe transcription a transcription start region and a translation startregion, the DNA sequence of interest and a transcription stop region andtranslation stop region that is functional in a microbial cell. Thetermination region may be native regarding the transcription initiationregion, may be native regarding the DNA sequence of interest or may bederived from any other source.

The expression “open reading frame” (ORF) refers to the amino acidsequence which is coded for between the translation start codons andtranslation stop codons of a coding sequence. The expressions “startcodon” and “stop codon” refer to a unit of three adjacent nucleotides(codons) in a coding sequence which specify the chain start and chainstop of the protein synthesis (mRNA translation).

In connection with a nucleic acid “operative linkage” refers to acompound as part of the same nucleic acid molecule in an appropriateposition and orientation to the transcription start of the promoter. DNAin functional connection to a promoter is located under thetranscription initiation regulation of the promoter. Coding sequencesmay be operatively linked with the regulator sequence in senseorientation or antisense orientation. Regarding polypeptides, operativelinkage refers to the connection as part of the same polypeptide, i.e.,via peptidyl bonds.

According to the invention any promoter may be used. Promoter usuallyrefers to the nucleotide sequence upstream (5′) to the coding sequenceand controls the expression of the coding sequence by providing therecognition of the RNA polymerase and other factors which are necessaryfor the correct transcription. The promoter used according to theinvention may comprise a minimal promoter, i.e., a short DNA sequencefrom a TATA box and other sequences which specify the transcriptionstart site to which regulator elements are attached for expressioncontrol.

The promoter used according to the invention may also comprise anucleotide sequence which comprises a minimal promoter and regulatorelements, and may control the expression of a coding sequence orfunctional RNA. This type of promoter sequence consists of proximal anddistal elements located upstream, whereby the elements named last areoften referred to as enhancers. Consequently, an enhancer is a DNAsequence which may stimulate the promoter activity and may be an elementinherent to the promoter or an inserted heterologous element to improvethe expression height or tissue specificity of a promoter. It may workin both orientations and may even work if it is located upstream ordownstream to the promoter. Not only enhancers but also other upstreamlocated promoter elements sequence-specifically bind DNA-bindingproteins mediating their effects. Promoters may be derived from a nativegene in their entirety or may be composed of different elements derivedfrom different naturally occurring promoters or may even be composed ofsynthetic DNA segments. A promoter may also comprise DNA sequences whichare involved in the binding of protein factors which control theefficiency of the transcription initiation as response to physiologicalor development-related conditions.

Promoter elements, particularly TATA elements which are inactive or havea strongly reduced promoter activity in the absence of an upstreamactivation are referred to as minimal promoters or core promoters. Inthe presence of an appropriate transcription factor or appropriatetranscription factors the function of the minimal promoter is theenabling of the transcription. Thus, a minimal promoter or core promoteronly consists of all basic elements which are necessary for thetranscription initiation, e.g., a TATA box and/or an initiator.

The invention also relates to vector constructs comprising DNA sequencesaccording to the invention. These vector constructs comprise anyplasmid, cosmid, phage or other vector in double-stranded orsingle-stranded, linear or circular form, which might be transmittableor mobilizable themselves and may either transform a prokaryotic oreukaryotic host by means of integration into the cellular genome or areextra-chromosomally present (e.g., autonomously replicating plasmidswith replication origin).

Vectors, plasmids, cosmids, artificial yeast chromosomes (YACs),artificial bacterial chromosomes (BACs) and DNA segments to be used forthe transformation of cells generally comprise the DNA which codes forthe phospholipase according to the invention as well as another DNA suchas cDNA, a gene or genes which is/are to be introduced into the cells.These DNA constructs may comprise further structures such as promoters,enhancers, polylinkers or also regulator genes, if necessary. One of theDNA segments or genes which was/were selected for the cellularintroduction conveniently codes/code for a protein which is expressed inthe thus obtained transformed (recombinant) cells, which leads to ascreenable or selectable property and/or provides the transformed cellwith an improved phenotype.

The construction of vectors which may be used according to the inventionis known to a person skilled in the art due to the aforementioneddisclosure and the general expert knowledge (cf., e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Plainview, N.Y. (1989))).

The expression cassette according to the invention may comprise one orseveral restriction site(s) to put the polynucleotide coding for thephospholipase under the control of a regulator sequence. The expressioncassette may also comprise a termination signal in operative linkagewith the polynucleotide as well as regulator sequences which arerequired for the proper translation of the polynucleotide. Theexpression cassette which comprises the polynucleotide according to theinvention may be chimeric, i.e., at least one of its components isheterologous in relation to at least one of the other components. Theexpression of the polynucleotide in the expression cassette may be underthe control of a constitutive promoter, an inducible promoter, aregulated promoter, a viral promoter or a synthetic promoter.

The vectors may already comprise regulator elements, e.g., promoters, orthe DNA sequences according to the invention may be manipulated in sucha way that they comprise such elements. Appropriate promoter elementswhich may be used are known in the state of the art and are, forexample, for Trichoderma reesei the cbh1 promoter or cbh2 promoter, forAspergillus oryzae the amy promoter, for Aspergillus niger the xylpromoter, glaA promoter, alcA promoter, aphA promoter, tpiA promoter,gpdA promoter, sucI promoter and pkiA promoter. Appropriate promoterelements which may be used for expression in yeast are known in thestate of the art and are, for example, the pho5 promoter or the gappromoter for expression in Saccharomyces cerevisiae and for Pichiapastoris, for example, the aoxI promoter or the fmd promoter, or the moxpromoter for H. polymorpha.

DNA which is appropriate for introduction into cells may also comprise,besides the DNA according to the invention, DNA which was derived orisolated from any source. An example of a derived DNA is a DNA sequencewhich was identified in a given organism as a useful fragment and thenchemically synthesized in a basically purified form. An example of sucha DNA is an appropriate DNA sequence which was, for example, obtained bythe use of restriction endonucleases, so that it may be furthermanipulated, for example, amplified according to the invention. The amdSgene from Aspergillus nidulans, which may be used as a marker gene, andits regulatory sequences as well as polylinkers are among those, interalia.

Such a DNA is usually referred to as recombinant DNA. Thus, anappropriate DNA comprises completely synthetic DNA, semi-synthetic DNA,DNA isolated from biological sources and DNA derived from introducedRNA. Generally, the introduced DNA is no original part of the genotypeof the recipient DNA, however, according to the invention, a gene mayalso be isolated from a given genotype and optionally altered andsubsequently multiple copies of the gene may be introduced into the samegenotype, e.g., to increase the production of a given gene product.

The introduced DNA comprises without limitation DNA from genes such asfrom bacteria, yeasts, fungi or viruses. The introduced DNA may comprisemodified or synthetic genes, parts of genes or chimeric genes includinggenes of the same or a different genotype. This may also include forexample DNA of the plasmids pUC18, pUC19.

The DNA used according to the invention for the transformation may becircular or linear, double-stranded or single-stranded. In general, theDNA is a chimeric DNA such as a plasmid DNA, which also comprises codingregions which are flanked by regulator sequences and support theexpression of the recombinant DNA present in the transformed cell. Forexample, the DNA itself may comprise or consist of a promoter that isactive in a cell, that is derived from a source differing from the cell,or a promoter that is already present in the cell, i.e., thetransformation target cell, may be used.

In general, the introduced DNA is relatively small, less than about 30kb, in order to minimize the sensitivity to physical, chemical orenzymatic degradation, which increases with the size of the DNA.

The selection of an appropriate expression vector depends on the hostcells. Yeast expression vectors or fungi expression vectors may comprisea replication origin, an appropriate promoter and enhancer as well asany necessary ribosome binding sites, polyadenylation sites, splicedonor sites and splice acceptor sites, transcription terminationsequences and non-transcribed 5′-flanking sequences.

Examples of appropriate host cells are: fungi cells of the genusAspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium etc.such as yeasts of the genera Kluyveromyces, Saccharomyces,Schizosaccharomyces, Trichosporon, Schwanniomyces, Hansenula, Pichia andthe like. Appropriate host systems are, for example, fungi such asAspergilli, e.g., Aspergillus niger (ATCC 9142) or Aspergillus ficuum(NRLL 3135), or Trichoderma (e.g., Trichoderma reesei QM6a) and yeastssuch as Saccharomyces, e.g., Saccharomyces cerevisiae, or Pichia suchas, e.g., Pichia pastoris, or Hansenula, e.g., H. polymorpha (DSMZ70277). Such microorganisms may be obtained from established depositaryinstitutions, e.g., the American Type Culture Collection (ATCC), theCentraalbureau voor Schimmelcultures (CBS) or the Deutsche Sammlung fürMikroorganismen and Zellkulturen GmbH (DSMZ) or any other depositaryinstitution.

The expression cassette may include, in the 5′-3′ transcriptiondirection, a transcription start region and translation start region ofthe polynucleotide according to the invention and a transcription regionand termination region that are functional in vivo or in vitro. Thetermination region may be native regarding the transcription initiationregion or may be native or of other origin regarding the polynucleotide.The regulator sequences may be located upstream (5′ non-codingsequences), inwardly (introns) or downstream (3′ non-coding sequences)of a coding sequence and may influence the transcription, the RNAprocessing or the stability and/or the translation of the associatedcoding sequence. Regulator sequences may comprise without limitationenhancers, promoters, repressor binding sites, translation leadersequences, introns or polyadenylation signal sequences. They maycomprise natural and synthetic sequences as well as sequences which area combination of synthetic and natural sequences.

The vector used according to the invention may also comprise appropriatesequences for the amplification of the expression.

Examples of promoters which may be used according to the invention arepromoters of which is known that they control the expression in theeukaryotic cells. Any promoter with the ability to express infilamentous fungi may be used. Examples are a promoter that is stronglyinduced by starch or cellulose, e.g., a promoter for glucoamylase orα-amylase from the genus Aspergillus or for cellulase(cellobiohydrolase) from the genus Trichoderma, a promoter for enzymesin the glycolytic metabolic pathway such as phosphoglycerate kinase(PGK) and glycerol aldehyde-3-phosphate dehydrogenase (GPD) etc. Thecellobiohydrolase-1 promoter, the cellobiohydrolase-II promoter, theamylase promoter, the glucoamylase promoter, the xylanase promoter orthe enolase promoter is preferred.

In addition to the use of a special promoter, other types of elementsmay influence the expression of transgenes. It was particularlydemonstrated that introns have the potential to amplify transgeneexpression.

The expression cassette may comprise further elements, for exampleelements which may be regulated by endogenous or exogenous elements suchas zinc finger proteins, including naturally occurring zinc fingerproteins or chimeric zinc finger proteins.

The expression cassette used according to the invention may alsocomprise enhancer elements or upstream promoter elements.

Vectors for the use according to the invention may be constructed insuch a way that they comprise an enhancer element. Thus, the constructsaccording to the invention comprise the gene of interest together with a3′ DNA sequence, which acts as a signal to terminate the transcriptionand to allow for the polyadenylation of the thus obtained mRNA. Anysignal sequence which makes the secretion from the selected hostorganism possible may be used. A preferred signal sequence is thephospholipase signal sequence from Aspergillus fumigatus or signalsequences derived therefrom for the secretion from filamentous fungi.

A special leader sequence may also be used, since the DNA sequencebetween the transcription start site and the start of the codingsequence, i.e., the non-translated leader sequence, may influence thegene expression. Preferred leader sequences comprise sequences whichcontrol the optimal expression of the adhered gene, i.e., they comprisea preferred consensus leader sequence, which increases or maintains themRNA stability and prevents an inappropriate translation initiation. Theselection of such sequences is well known to a person skilled in theart.

In order to improve the possibility to identify the transformants, aselectable or screenable marker gene may be included in the expressioncassette. Such marker genes are well known to a person skilled in theart.

The expression cassette or a vector construct which comprises theexpression cassette is introduced into a host cell. A variety oftechniques is available and well known to a person skilled in the art ofintroducing constructs into a host cell. The transformation of microbialcells may be carried out by means of polyethylene glycol, calciumchloride, viral infection, DEAE dextran, phage infections,electroporation and other methods known in the state of the art. Thetransformation of fungi may be carried out according to Penttila et al.,Gene 61:155-164, 1987. The introduction of a recombinant vector intoyeasts may be carried out according to methods known per se, includingelectroporation, use of spheroplasts, lithium acetate and the like.

As soon as the expression cassette or the DNA sequence according to theinvention is obtained, it may be introduced into vectors according toprocesses known per se in order to over-express the encoded polypeptidein appropriate host systems. However, DNA sequences as such may also beused to transform appropriate host systems of the invention in order toobtain an overexpression of the encoded polypeptide.

As soon as a DNA sequence according to the invention is expressed in anappropriate host cell in an appropriate medium, the encodedphospholipase may be concentrated and/or isolated according to processesknown per se either from the medium, if the phospholipase is secretedinto the medium, or from the host organism, if the phospholipase isintracellularly present, e.g., in the periplasmic space. Known processesfor the separation of the insoluble parts of the culture medium and thebiomass followed by processes for concentrating the phospholipase may beused to produce concentrated phospholipase solutions or to prepare thedrying of the phospholipase. For example, filtration processes orcentrifugation processes may be used for the removal of the insolublecomponents followed by ultrafiltration processes for concentration, orcross flow filtration processes are used. Drying may be carried out bymeans of freeze or spray drying, granulation processes, extrusion orother processes. Known processes of protein purification may be used toisolate the phospholipases according to the invention. For example,various chromatographic or gel chromatographic processes may be usedindividually or in combination. Depending on the host cell used in arecombinant production process, the enzyme according to the inventionmay or may not be covalently modified by means of glycosylation. Ineukaryotic cells the glycosylation of the secreted proteins provides abasis for the modulation of the protein folding, the conformationstability, the thermal stability and the resistance against proteolysis.As regards a specific use of the phospholipase, a glycosylated variantof the enzyme may be preferred to a non-glycosylated variant.

The invention also relates to isolated or basically purified nucleicacid compositions and protein compositions. An isolated and purifiedpolynucleotide/polypeptide or segment thereof refers to a polynucleotideor polypeptide or segment thereof which is isolated from its nativeenvironment and is present in a purified form for further use. Anisolated polynucleic acid segment or polypeptide may be present in apurified form or may be present in a non-native environment such as in atransgenic host cell. For example, an isolated or purifiedpolynucleotide segment or protein or a biologically active part thereofis basically free of further cellular material or culture medium ifproduced according to recombinant techniques or is basically free ofchemical precursors or other chemical compounds. An isolatedpolynucleotide is preferably free of sequences (preferablyprotein-encoding sequences) which naturally flank the nucleic acid(i.e., sequences which are localized at the 5′ end and 3′ end of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, according to different embodiments, theisolated nucleic acid molecule may comprise less than approximately 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequenceswhich naturally flank the nucleic acid molecule in the genomic DNA ofthe cell from which the nucleic acid is derived. A protein that isbasically free of cellular material comprises compositions of proteinand polypeptide with less than approximately 70%, 50%, 30%, 20%, 10%, 5%(in relation to the dry weight) of contaminating protein. If the proteinaccording to the invention or a biologically active fragment thereof isrecombinantly produced, the culture medium preferably comprises lessthan approximately 70%, 50%, 30%, 20%, 10% or 5% (in relation to the dryweight) of the chemical precursors or non-protein-like chemicalsubstances.

The invention also relates to phospholipase compositions which comprisethe polypeptide according to the invention. Phospholipase compositionsare generally liquid or dry. Liquid compositions preferably comprise thephospholipase enzyme in a purified or enriched form. However, auxiliaryagents such as a stabilizer and/or glycerol, sorbitol or monopropyleneglycol, additives such as salts, sugar, preservatives, agents to adjustthe pH value and proteins may be added. Typical liquid compositions areaqueous or oily suspensions.

Dry compositions may be freeze-dried, spray-dried, granulated orextruded compositions, which may only comprise the enzyme. Drycompositions may be granulates which may easily be mixed with, forexample, food or, feed components, or which preferably form a componentof a premix. Preferably, the particle size of the enzyme granulate iscompatible with that of the other component of the mixture. This allowsfor safe and purposeful agents to incorporate enzymes in processed food,premixes or animal feed, for example.

Dry compositions may also comprise other additives such as salts,particularly phosphate salts and their anhydrous forms, and stabilizerssuch as poly(vinyl pyrrolidone) etc. to regulate certain conditions suchas the pH value in the application.

A food additive according to this embodiment of the present inventionmay be combined with other food components in a similar way, wherebyprocessed food products are produced. Such other food componentscomprise one or more enzyme supplements, vitamins, minerals or traceelements. The thus obtained combined dietary supplement may then bemixed with other food components such as grain and plant proteins in anappropriate amount to obtain processed food. The processing of thesecomponents to processed food may be carried out by means of processingdevices known per se.

In a preferred embodiment the phospholipase compositions according tothe invention additionally comprise an effective amount of one or moreenzyme(s) for food or animal feed or for the application in pre-stagesfor the production of food or animal feed or for the application intextile industry, preferably selected from alpha-galactosidases,beta-galactosidases, laccases, other phospholipases, phosphatases,endoglucanases, particularly endo-beta-1,4-glucanases,endo-beta-1,3(4)-glucanases, endo-1,2-beta-glucanases andendo-1,3-alpha-glucanases, cellulases, xylosidases, galactanases,particularly arabinogalactan-endo-1,4-beta-galactosidases andarabinogalactan-endo-1,3-beta-galactosidases, pectin-degrading enzymes,particularly pectinases, pectinesterases, pectinlyases,polygalacturonases, arabananases, rhamnogalacturonases,rhamnogalacturonanacetylesterases,rhamnogalacturonan-alpha-rhamnosidases, pectate lyases andalpha-galacturonidases, mannanases, beta-mannosidases, mannanacetylesterases, xylan acetylesterases, proteases, xylanases,arabinoxylanases, lipolytic enzymes such as lipases,digalactosid-diglycerol esterases and cutinases, and other enzymes suchas laccases and transglutaminases.

The phospholipases according to the invention may be used for a varietyof applications. Examples are applications in baking and in animalfeeding as well as in the production of fuels from renewable energysources such as rapeseed, or in the processing of textile raw materials.

A preferred application is the use of the polypeptides havingphospholipase activity according to the invention in processes for thedegumming of vegetable oil. The edible oil to be degummed is, forexample, treated with a polypeptide according to the invention, wherebythe majority of the phospholipids is hydrolyzed, and subsequently theaqueous phase containing the hydrolyzed phospholipids is separated fromthe oil. Such a process is particularly suitable for the purification ofedible oils containing phospholipids, for example, vegetable oils suchas soy bean oil, rapeseed oil and sunflower oil.

Prior to the phospholipase treatment, the oil is preferably pre-treatedto remove gummy substances, for example, by humid refining. Typically,the oil comprises 50 to 850 ppm phosphorus as phospholipid at thebeginning of the treatment with the phospholipase according to theinvention. After the treatment, the phosphorus content is typicallybetween 2 and 10 ppm.

The phospholipase treatment is generally carried out in such a way thatthe phospholipase is dispersed in an aqueous solution, preferably in theform of droplets of an average diameter of <10 μm. The amount of wateris preferably 0.5 to 5 wt. % (w/w) in relation to the oil. An emulsifiermay optionally be added. It may be mechanically stirred to maintain anemulsion. The treatment with phospholipase may be carried out at a pHvalue in the range of approximately 3.5 to approximately 5.0. The pHvalue of the process may be in the range of approximately 3.5 toapproximately 5, preferably 3.8 to 4.5 and most preferably 4.0 to 4.2 tomaximize the performance of the enzyme. The pH value may be adjusted by,e.g., addition of citric acid, a citrate buffer, phosphoric acid orhydrochloric acid. An appropriate temperature is generally 30°-70° C.,preferably 45°-65° C. and most preferably 55°-62° C. The reaction timeis typically 1 to 12 hours, preferably 2 to 6 hours. An appropriateenzyme dosage is usually 120 to 3,000 units per kg oil, preferably 250to 2,000 and most preferably 750 to 1,500 units per kg oil.

The phospholipase treatment may be carried out batchwise, for example ina tank under stirring, or may be continuous, for example, in a series oftank reactors under stirring.

The phospholipase treatment is followed by the separation of an aqueousphase and an oily phase. The separation may be carried out byconventional means, for example, centrifugation. The aqueous solutioncontains phospholipases, and the enzyme may be used again to improve theeconomy of the process.

The treatment may be carried out by means of processes known per se.

Advantageously, the phospholipase according to the invention may also beused to prepare dough and bakery products, whereby an effective amountof a polypeptide according to the invention is worked into the dough. Byadding a polypeptide having phospholipase activity according to theinvention, one or several property(ies) of the dough or the bakeryproduct prepared by means of the dough may be improved compared to adough or bakery product to which no polypeptide having phospholipaseactivity according to the invention was added.

In the dough preparation by means of the phospholipase according to theinvention the phospholipase may be added to the dough itself, to anyingredient of which the dough is prepared, and/or to a mixture of doughingredients of which the dough is prepared. A polypeptide havingphospholipase activity according to the invention may thus be added assuch in any step of the dough preparation or may be added in one, two ormore step(s). Here an effective amount is to refer to an amount ofphospholipase that is sufficient to cause a measurable effect on atleast one property of interest of the dough and/or the bakery product.

The expression “improved property” is defined herein as any property ofthe dough and/or the product that is obtained from the dough,particularly a bakery product, which was improved due to the effect ofthe phospholipase in relation to the dough or the product to which thephospholipase according to the invention was not added. The improvedproperty may comprise, for example: improved dough strength, improvedelasticity of the dough, improved stability of the dough, reducedstickiness of the dough, improved extensibility of the dough, improvedmachinability of the dough, increased volume of the bakery product,improved crumb structure of the bakery product, improved softness of thebakery product, improved aroma of the bakery product and/or delayedstaling of the bakery product. Processes to determine these propertiesare well known in the state of the art.

Dough is herein defined as a mixture of flour and other ingredients,which is solid enough to be kneaded or rolled. The dough may be fresh,frozen, precooked or pre-baked.

The expression “bakery product” herein refers to any product which isprepared by dough and is either soft or crisp. Examples of bakeryproducts which may be prepared by means of a phospholipase according tothe invention are, for example, bread (particularly white bread,wholemeal bread or rye bread), typically in the form of loaves or Frenchbread of the type French baguette, pasta, pita bread, tortillas, tacos,cakes, pancakes, cookies and pastries, cooked bread, double-baked breadand the like.

In the preparation of these bakery products the polypeptide havingphospholipase activity according to the invention and/or one or morefurther enzyme(s) in any formulation that is suitable for the respectiveuse may be added, for example, in a dry form, as a liquid or as apremix. Furthermore, one or more further enzyme(s) may be added to thedough. These further enzymes may be of any origin and may be derivedfrom mammals and plants, for example. Preferably, they are of amicrobial origin and are particularly preferably derived from bacteriaor fungi.

According to a preferred embodiment, the further enzymes may be amylasessuch as α-amylase (suitable for producing sugars that are fermentable byyeasts and for delaying staling) or 3-amylase, cylcodextringlucanotransferase, peptidase, particularly an exopeptidase (suitablefor increasing the aroma), transglutaminase, lipase (suitable formodification of the lipids present in the dough or in parts of the doughto make the dough softer), phospholipase (useful for modification of thelipids that are present in the dough or in parts of the dough to makethe dough softer and to improve the gas retention in the dough),cellulase, hemicellulase, particularly a pentosanase such as xylanase(useful for the partial hydrolysis of pentosanes which improve theextensibility of the dough), proteases (useful for gluten softening,particularly if durum flour is used), protein disulphid isomerase (forexample, a protein disulphid isomerase disclosed in WO 95/00636),glycosyl transferase, peroxidase (useful for improving the consistencyof the dough), laccase or oxidase, for example, an aldose oxidase,glucose oxidase, pyranose oxidase, lipoxy genase or L-amino acid oxidase(useful for improving the consistency of the dough).

This/These optionally further added enzyme/enzymes may be optionallyadded separately or together with the polypeptide having phospholipaseactivity according to the invention as components of baking agents ordough additives. The invention also relates to the preparation of suchdoughs as well as to the preparation of corresponding bakery productsmade of these doughs.

The invention also relates to a premix, for example, in the form of aflour composition, for the preparation of dough and/or bakery productsmade of dough, whereby this premix comprises polypeptides havingphospholipase activity according to the invention.

The polypeptides having phospholipase activity according to theinvention may also be used as additives to animal feed. Addingphospholipases to feed improves the efficiency of feed conversion ofanimals. Thus the growth of animals which are nourished with such feedis improved. A phospholipase according to the invention may hereby beadded as such or as feed concentrate. Furthermore, the phospholipase mayalso be added to the animal feed via transgenic plants, wherein thephospholipase was synthesized by heterologous gene expression. Processesfor the production of such transgenic plants are disclosed in EP0449376:

The polypeptides having phospholipase activity according to theinvention may also be used in the process of scouring in the processingof textile raw materials such as cotton fibres to facilitate the furthertreatment of the fibres. The improvements obtained by scouring influencethe behavior during staining as well as the further mechanic andenzymatic processing of the fibre and the fabric made thereof.

The gene for the phospholipase which was isolated from the microorganismAspergillus fumigatus was deposited in the plasmid pPL3949-Topo2.5 underaccession number DSM 22741 at the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSMZ), Inhoffenstraβe 7B, D-38124 Braunschweig onJul. 2, 2009 in accordance with the provisions of the Budapest Treaty.

The invention is further described on the basis of the enclosed figures.It is shown in:

FIG. 1: IEF gel of purified phospholipase from Aspergillus fumigatus.

-   -   Track 1: marker protein from the Isoelectric Focusing        Calibration Kit, pH 2.5-6.5,    -   Track 2-3: The phospholipase band at pI approx. 4.1 is        identified by an arrow.

FIG. 2: T optimum curve for the recombinant phospholipase expressed inTrichoderma reesei RH32664.

FIG. 3: pH optima curve for the recombinant phospholipase expressed inTrichoderma reesei RH32664.

FIG. 4A and FIG. 4B: Nucleotide sequence and amino acid sequence derivedtherefrom of the chromosomal phospholipase gene from Aspergillusfumigatus RH3949. The introns are printed in italics and the amino acidsequence in bold face type (nucleotide sequence disclosed as SEQ ID NO:1, amino acid sequence disclosed as SEQ ID NO: 2).

FIG. 5: The nucleotide sequence of the chromosomal phospholipase genefrom Aspergillus fumigatus RH3949 (SEQ ID NO: 1).

FIG. 6: The amino acid sequence of the phospholipase gene fromAspergillus fumigatus RH3949 (SEQ ID NO: 2).

FIG. 7: Restriction map of the vector pPL3949-Topo2.5

FIG. 8: Restriction map of the expression vector pAB500-PL3949

REFERENCE EXAMPLE 1 Determination of the Phospholipase Activity

1 phospholipase unit (PLU) corresponds to the amount of enzyme whichreleases 1 μmol fatty acid per minute from the phosphatidyl cholineunder standard conditions.

Reagents: Substrate Emulsion:

1 g Epikuron 200 (purified phosphatidyl choline derived from soy of thecompany Lucas Meyer, now available at Cargill), 100 ml deionized waterand 5 ml 0.32 M CaCl₂ solution are homogenized by means of an UltraTurrax for 2 min at 24,000 rpm. The substrate emulsion is stable at4°-8° C. for 3-4 d.

Other Solutions:

0.32 MgCl₂ solution, fresh 3.3 mM citric acid-monohydrate solution, 10mM KOH solution, 1% Triton X100 (company Fluka) solution indemineralized water

Enzyme Solution:

The enzyme preparations are dissolved in deionized water. The enzymeconcentration in the batch may not exceed 2.5 U g⁻¹.

Procedure:

Main values

-   -   10 ml substrate emulsion    -   10 ml 1% Triton X100 solution    -   5 ml 3.3 mM citric acid-monohydrate solution        were pipetted in a 25 ml wide-necked Erlenmeyer flask and        tempered at 40° C. for 10 min. The pH value adjusts to        approximately 3.3-3.5.

After adding 0.1 ml of enzyme solution, the analysis batch was incubatedfor 10 min at 40° C. When the incubation time is over, the solution istitrated with 10 mM KOH to pH 10.0, whereby the first 5 ml of KOH areadded rapidly (duration: about 1 min). The consumption of KOH isregistered.

Blank Values

The enzyme parent solution is heated for 15 min at 95° C. and is thusdeactivated. After cooling down to room temperature, the furthertreatment is the same as for the main values.

An incubation of the blank values is not necessary.

Evaluation:

${{PLU}\text{/}g} = \frac{\Delta \; V_{KOH}*_{CKOH}*1000}{\Delta \; t*c_{s}*v}$

-   V_(KOH) (ml) difference in consumption between the blank value and    the main value-   c_(KOH) (mol l⁻¹) concentration of the KOH solution-   Δt (min) incubation time-   c_(s) (g ml⁻¹) concentration of the sample-   v (ml) volume used

REFERENCE EXAMPLE 2 Rapid Test for the Detection of Phospholipase at pH3.5 Reagents: Substrate Emulsion:

1 g Epikuron-200 is mixed with 100 mg Milli Q Water and 5 ml 0.32 Mcalcium chloride solution and is homogenized by means of theUltra-Turrax (for about 1-2 min at about 24000 rpm).

For the analysis batch 10 ml of this substrate emulsion are mixed with10 ml of 1% Triton X-100 solution and 5 ml of 3.3 mM citric acidsolution-monohydrate solution.

Free Fatty Acids, Semi-Micro Test (Boehringer)

(The semi-micro test contains the reagents for the reaction mixture A,reaction mixture B and the N-ethylmaleimide).

Procedure:

At first 5 μl diluted enzyme solution are provided in the microtiterplate and mixed with 0.1 ml substrate emulsion.

The substrate/enzyme batches are incubated in a water bath for 10 min at40° C.

Subsequently, 5 μl of the batch are pipetted into a second microtiterplate to 100 μl of the reaction mixture A and are incubated for 5 min at40° C.

After the incubation time is over 5 μl of a mixture of reaction mixtureB and N-ethylmaleimide solution at a ratio of 1:1 are added and againincubated for 5 min at 40° C.

Phospholipase activity is present if the reaction batch gets a redcolor.

REFERENCE EXAMPLE 3 Determination of the Content of Free Fatty AcidsReagents:

Ethanol (96% solution), toluol

Ethanol and toluol are mixed in the ratio 1:1 (v/v).

0.1 N ethanolic KOH

1% phenolphthalein solution in ethanol

Procedure:

Approximately 3 g water-free oil are weighed out into an Erlenmeyerflask to four decimal places exactly, dissolved in 20 ml ethanol-toluolmixture, mixed with 2 to 3 drops of phenolphthalein and titrated with0.1 N KOH solution until it has a lasting red color.

Evaluation:

The acid number is an indicator for the content of free fatty acids. Theacid number refers to the amount of potassium hydroxide in g necessaryfor the neutralisation of the free fatty acids contained in 1 kg oil.The acid number (AN) is calculated according to the following equation:

${AN} = \frac{a*N*56.1}{E}$

a amount of KOH solution in ml usedN normality of KOHE amount of fat in g weighed out56.1 molar mass of KOH in g/mol

The acid number may be used to calculate the content of free fatty acids(FFA) in percent:

${{FFA}\mspace{14mu} (\%)} = {{AN}*\frac{282*100}{56.1*1000}}$

282 molar mass of the oleic acid

REFERENCE EXAMPLE 4 Detection of Lipase

The qualitative detection of lipase on olive oil-agar is analogouslycarried out according to the method of Kouker and Jaeger (AppliedEnviron. Microbiol., 59: 211-213 (1987)).

For the detection of lipase activity agar plates made of tributyrin agar(Fluka 91015) which were produced with 1% olive oil are used. The pHvalue adjusts to 5.5.

The detection of lipase activity is photometrically carried out withemulsified p-nitrophenylpalmitate (Sigma N2752) as a substrate in 0.5 Mcitrate/phosphate buffer, pH 5.1 according to Winkler and Stuckmann(1979) (J. Bac., 138:663-670 (1979)).

EXAMPLE 1 Extraction of Phospholipase from Aspergillus fumigatus StrainRH3949

Aspergillus fumigatus was grown in 200 ml shaking flasks filled with 50ml medium at 28° C., 200 rpm, over 5 d. The medium consisted of 0.5%Epicuron 200 (Lucas Meyer), 0.5% corn steep powder, 0.2% NH₄NO₃, 100 mMKH₂PO₄ and 0.1% Triton X100. The pH value was adjusted to pH 6 beforesterilisation. The medium was inoculated with a spore suspension. After5 days the culture supernatant was separated from the mycelium by meansof filtration and the phospholipase activity in the liquid was measured.

EXAMPLE 2 Purification of the Phospholipase from Aspergillus fumigatusStrain RH3949 Step 1: Anion Exchanger, Macro Prep Q

In order to purify the phospholipase at first a separation of proteinswas carried out at the anion exchanger Macro Prep Q.

To this end the concentrated culture supernatant from the culturesaccording to example 1 was diluted with completely desalinated wateruntil the protein solution had the same conductivity as buffer A.Subsequently, the protein sample was adjusted to pH 7 with 1 M NaOH andloaded onto the column equilibrated with buffer A. After washing thecolumn with buffer A the phospholipase was eluted with a linearlyincreasing NaCl gradient of 0-1 M. The fractions having phospholipaseactivity were combined and further purified.

Buffer A: 5 mM CaCl₂+20 mM Tris-HCl, pH 7.0 Buffer B: 5 mM CaCl₂+20 mMTris-HCl, pH 7.0+1 M NaCl Step 2: HIC, Phenyl Sepharose 6 Fast Flow LowSubstitution

The protein sample having phospholipase activity according to step 1 wasmixed with 3.4 M ammonium sulphate solution at a ratio of 1:1 andadjusted to pH 7.0 with 1 M NaOH solution. After applying the sample tothe phenyl-sepharose column, which was also equilibrated with buffer A,the phospholipase was eluted with a decreasing ammonium sulphategradient.

Buffer A: 5 mM CaCl₂+20 mM Tris-HCl, pH 7.0+1.7 M ammonium sulphate

Buffer B: 5 mM CaCl₂+20 mM Tris-HCl, pH 7.0 Step 3: Gel Filtration,Superose 12 HR 10/30

As a final purification step the proteins were separated at a gelfiltration column. To this end the phospholipase sample according tostep 2 was dialyzed against completely desalted water in a dialyse tube(Naturin protein farce) for 1.5 h and subsequently lyophilised. Thelyophilisate was absorbed in 500 μl completely desalinated water. 250 μlwere applied to the column in two steps, respectively, and eluted withbuffer A.

Buffer A: 5 mM CaCl₂+20 mM Tris-HCl, pH 7.0

The purified phospholipase was applied to an IEF gel. The result isdisplayed in FIG. 1.

The bands were cut out for identification and examined for phospholipaseactivity according to the described method of analysis.

EXAMPLE 3 N-Terminal Protein Sequencing

After the final purification step via the superose the purified proteinwas separated on a native gel. The protein bands having phospholipaseactivity were cut out and again applied to an SDS gel in order todetermine the molecular weight. For the determination of N-terminalamino acids the protein bands were transferred from the native gel to aPVDF membrane (Fluotrans Transfer Membrane, Pall) and the N-terminalamino acid sequences were determined in an amino acid sequencer (AppliedBiosystems Model 470A) according to the Coomassie staining process. Theyare:

¹DVSAS VLQKL SLFAQ Y¹⁶ (SEQ ID NO: 3)

Comparing the sequences shows that the N-terminal amino acid sequence ofthe phospholipase gene has a high sequence similarity with the gene ofthe extracellular lipase from the Aspergillus fumigatus strain Af293(GenBank EAL86100).

EXAMPLE 4 Cloning of the Chromosomal Phospholipase Gene from theAspergillus fumigatus Strain RH3949 by Means of Polymerase ChainReaction (PCR)

Different oligoprimers for the amplification of the phospholipase DNAwere derived from the data of the chromosomal DNA sequence of theAspergillus fumigatus lipase. The chromosomal DNA preparation wascarried out according to modified instructions of Hynes, M. J et al.,(1983) Mol. Cell. Biol. 3, 1430-1439. The amplification of thephospholipase gene was carried out by means of the PCR method. The PCRproducts were cloned in pCR2.1-TOPO plasmid and sequenced. Hereby it isshown that the primer pair N2-3948 and 3949-ApaI leads to the genehaving the phospholipase DNA according to the invention.

N2-3948 (SEQ ID NO. 4) 5′-AGAGTCTGCC TATATTCTCT CTGAAAGG-3′ 3949-ApaI(SEQ ID NO: 5) 5′-TATGACAATTTCCGTGATTACTG-3′

The reaction batch of 100 μl comprised: 10 μl 10× buffer (200 mMTris/HCl, pH 8.4, 500 mM KCl), 3 μl 50 mM MgCl₂, 2 μl 10 mM dNTP, 50pMol oligoprimer (N2-3948 and 3949-ApaI) each, approx. 10 ng chromosomalDNA, 5 U Taq DNA polymerase (Invitrogen). The batch was treated fordenaturation at 95° C./5 min, 45 cycles (95° C./1 min, 45° C./1 min, 72°C./1 min) and the subsequent extension was carried out at 72° C./10 min.

The PCR products were purified on a Qiaquick column and cloned inpCR2.1-TOPO plasmid. After sequencing one transformant comprised thephospholipase DNA sequence according to the invention (FIG. 5, SEQ IDNO: 1) and was referred to as pPL3949-Topo2.5 (FIG. 7).

The open reading frame which codes for the phospholipase comprises 1052nucleotides containing 299 amino acids. The Phospholipase gene comprises3 introns.

The derived N-terminal amino acid sequence corresponds to the peptidesequences determined by means of protein sequencing (Example 3, SEQ IDNO: 3).

The derived molecular weight of approx. 28.6 kDa corresponds to theapprox. 29 kDa which were determined by means of SDS-PAGE (Example 8).

The determination of the signal sequence was carried out with the helpof a computer program (PSORT) by Nakai and Kanehisa (1992, Genomics 14,897-911). According to this program the phospholipase gene has a signalsequence of 21 amino acids and a propeptide of 8 amino acids.

EXAMPLE 5 Construction of the Expression Vector pAB500-PL3949

In the expression vector pAB500-PL3949 the phospholipase gene is undercontrol of the T. reesei cbhI promoter and cbhI terminator.

For the construction of the plasmid pAB500-PL3949 the gene coding forphospholipase was amplified from the plasmid pPL3949.Topo2.5 by means ofPCR. The PCR product was hydrolysed with the enzymes AvrII/PacI andsubsequently inserted in the SpeI and PacI cleavage sites after the T.reesei cbh1 promoter in the plasmid pAB500. The plasmid obtained isreferred to as pAB500-PL3949.

The construction of the plasmid pAB500 was carried out according to thefollowing steps:

By introducing further cleavage sites (SpeI and PacI in the SacII sitebetween the cbhI promoter and the cbhI terminator) the plasmid pAB487was produced from the plasmid pALK487 (WO94/28117). The SpeI-Pactcleavage sites are used for the direct cloning of the phospholipasegene.

The amdS gene including its promoter and its terminator was amplifiedfrom the plasmid p3SR2 (GenBank 16371) by means of PCR. The PCR productwas cut with AscI and NruI and inserted in the AscI/StuI cleavage siteof pAB487, whereby the plasmid pAB500 was obtained.

EXAMPLE 6 Transformation of T. reesei

The techniques used for the transformation and treatment of T. reeseiwere those according to Penttila et al. (1987, Gene 61: 155-164).

T. reesei RH32439 was transformed with the linearized expressioncassette isolated from the plasmid pAB500-PL3949.

The transformants were selected and purified by means of single sporeisolation. Of all transformants, those having the highest secretioncapacity were chosen and further used in Example 7 for the production ofenzyme material.

EXAMPLE 7 Production of Enzyme Solutions by Means of Fermentation inShaking Flasks

Transformants which carry the expression cassette of Example 6 werecultivated on a cellulase-induced medium in shaking flasks. The culturefiltrates obtained after 6 days of growing were used for thecharacterization of the enzyme (Example 8) and for the analysis of theoil degumming process (Example 9).

EXAMPLE 8 Characterization of the Recombinant Phospholipases

The determination of the molecular weight by means of SDS polyacrylamidegel electrophoresis (SOS-PAGE) resulted in a molecular weight of approx.29 kDa for the phospholipase according to the invention.

The N-terminal sequencing of the recombinant phospholipase was carriedout by the company Chromatec (Germany). The amino acid sequence obtainedby means of sequencing corresponds to the N-terminal sequence of thephospholipase (Example 3).

The identification of the recombinant protein by means of MALDI-MS wascarried out by the company Protagen (Germany). The result confirms thatthe protein sequence of the phospholipase according to the invention iscorrect.

The temperature dependency of the enzyme activity was determined atdifferent temperatures by means of the determination method as describedabove. The temperature optimum of the phospholipase is 50° C. (cf. FIG.2).

The optimal pH value of the enzyme activity was determined at differentpH values by means of the determination method as described above. Tothis end the pH value in the reaction batch was adjusted with the helpof citric acid.

The enzyme is active in a broad pH range of pH 3 to 5 (cf. FIG. 3).

The lipase activity was determined by means of the determination methoddescribed above. The A. fumigatus phospholipase according to theinvention has a very low lipase activity. According to the calculationsthe ratio of phospholipase activity to lipase activity was 7480:1 (withan experimental variation limit of ±10%).

EXAMPLE 9 Degumming of Oil by Means of an Enzyme from CultureSupernatants of Recombinant Trichoderma reesei Strains Having the Genefrom A. fumigatus RH3949

200 g edible oil (soybean oil 1 with a phosphorus content of 163.6 ppm;rapeseed oil with a phosphorus content of 161.6 ppm; soybean oil 2 witha phosphorus content of 592.8 ppm and soybean oil 3 with a phosphoruscontent of 81.1 ppm) were mixed with 0.42 ml of a 46% citric acidsolution and water in a 400 ml beaker glass. Thereby the total watercontent should not exceed 2%. The pH value of the reaction mixture wasadjusted to pH 4.0 by adding a 7% NaOH solution (0.6 ml). After addingthe enzyme solution according to the invention (50 U) the reaction batchwas mixed with the help of an Ultra Turrax for 2 min at 24000 rpm.Subsequently, the mixture was filled into a three-necked round-bottomedflask and incubated at 55° C. or 60° C. while stirring gently (200 rpm).

A sample of 20 ml was taken each 120 min after addition of the enzymesolution. The samples were centrifuged for 5 min at 4300×g and thephospholipide content, shown in ppm phosphorus, was photometricallydetermined as phosphorus molybdate complex at 830 nm in the oil afterashing at 850° C. by adding magnesium oxide.

The recombinant Trichoderma reesei strain which comprises the plasmidpAB500-PL3949 according to the invention is referred to as RH32664.

The results obtained in the process of degumming edible oil at 55° C. or60° C. by means of a phospholipase derived from a recombinant strain ofAspergillus fumigatus RH3949 are shown in Table 2.

The results show a clear degumming effect in the degumming process usingthe enzyme according to the invention compared to the water degummingprocess using citric acid. After only 4 hours the content of theremaining phosphorus is less than 10 ppm.

TABLE 2 Degumming of soybean oil and rapeseed oil by means ofphospholipase enzymes according to the invention (250 U per kg of edibleoil) from culture supernatants of a recombinant Trichoderma reeseistrain having a water content of 2% at pH 4.0 phosphorus content (ppm)time temp (° C.) name of sample (min) 55 60 citric acid 240 109.7 15.514.9 360 117.7 13.4 15.3 RH32664 240 8.4 4.1 4.6 (phospholipase) 360 7.82.4 2.0 soybean oil 1 untreated 163.3 rapeseed oil untreated 161.6soybean oil 2 untreated 592.8

EXAMPLE 10 Content of Free Fatty Acids in the Oil Degumming Process

The determination of the content of free fatty acids in the oildegumming process was carried out as described in reference example 3.The edible oil was mixed with phospholipase as described in Example 9and incubated for 6 hours at 57° C. The enzymatic hydrolysis ofphospholipids was caused by phospholipases which cleave the fatty acid.For reasons of comparison the same analysis was carried out with pureedible oils as blank value.

In contrast to the blank value (BV) the content of free fatty acid (FFA)in the samples rises only slightly and the difference (Δ_(FFA)) is 0.18%after treating the edible oil with phospholipase at a temperature of 57°C. (Table 3).

TABLE 3 Content of free fatty acids after treating the oil withphospholipase name of time phosphorus FFA Δ_(FFA) sample (min) content(ppm) (%) (%) citric acid 240 37.2 360 42.4 0.09 RH32664 240 2.0(phospholipase) 360 3.0 0.27 0.18 soybean oil 3, 81.1 untreated

1.-6. (canceled)
 7. A polypeptide having phospholipase activity essentially without lipase activity selected from a) a polypeptide having the sequence according to SEQ ID NO: 2 or a sequence derived therefrom, which may be obtained by substitution, addition, or deletion of one or more amino acid(s), b) a polypeptide having a sequence that has at least 83% identity with the amino acids 1 to 299 of SEQ ID NO: 2, c) a polypeptide which is coded for by a nucleic acid sequence which hybridizes under stringent conditions with (i) nucleotides 55 to 1106 of SEQ ID NO: 1, (ii) the cDNA sequence contained in nucleotides 55 to 1106 of SEQ ID NO: 1, (iii) a partial sequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii) or (iii), d) a variant of the polypeptide having SEQ ID NO: 2, comprising a substitution, deletion and/or insertion of one or more amino acid(s), e) allelic variants to amino acid sequences a) to e).
 8. A polypeptide having phospholipase activity characterized in that it has a molecular weight in the range of 28 to 30 kDa may hydrolyze at least one of the two fatty acids of lecithin essentially shows no lipase activity has a high temperature resistance may be isolated from an organism of the genus Aspergillus.
 9. The polypeptide according to claim 8 characterized in that it may be isolated from Aspergillus fumigatus.
 10. The polypeptide according to claim 9 characterized in that it is immunologically reactive with an antibody directed against the sequence SEQ ID NO:
 2. 11. The polypeptide according to any one of claims 8 to 10 characterized in that it comprises the sequence according to SEQ ID NO:
 2. 12. A phospholipase composition characterized in that it comprises a polypeptide according to any one of claims 7 to 11 together with additives and that it optionally further comprises one or more enzyme(s) for food or animal feed.
 13. The use of a polypeptide according to any one of claims 7 to 11 or a phospholipase composition according to claim 12 for the degumming of vegetable oil, for the preparation of dough and/or bakery products, for the preparation of dairy products, as additive to animal feed or for the processing of textile raw materials.
 14. (canceled)
 15. The process for degumming of vegetable oil characterized in that a) the vegetable oil to be treated is optionally pre-treated, b) an aqueous solution containing a polypeptide according to any one of claims 7 to 11 or a phospholipase composition according to claim 12 is added to the thus optionally pre-treated oil, c) the reaction batch according to b) is heated 1 to 12 h to a temperature of between 30° C. and 70° C. and d) the aqueous and oily phases are separated.
 16. A polypeptide with phospholipase activity essentially without lipase activity comprising a polypeptide which is encoded for by the coding part of a DNA sequence selected from the group consisting of a) DNA sequences that comprise a nucleotide sequence according to SEQ ID NO: 1, b) DNA sequences that comprise the coding sequence according to SEQ ID NO: 1, c) DNA sequences that code for the protein sequence according to SEQ ID NO: 2, d) DNA sequences that are coded for by the plasmid pPL3940-Topo2.5 with the restriction map according to FIG. 7, which is deposited under accession number DSM 22741, e) DNA sequences that hybridize under stringent conditions with one of the DNA sequences according to a), b), c) or d), wherein said DNA sequences hybridize at 65° C., 18 h in dextran sulphate solution, subsequent washing of the filter for 30 min, respectively, at first with 6× saline-sodium citrate (SSC), twice 2×SSC, twice 2×SSC, 0.1% SDS, and with 0.2×SSC at 65° C., and f) complementary strands to the sequences according to a) to f), wherein the DNA sequence is derived from Aspergillus.
 17. A polypeptide with phospholipase activity essentially without lipase activity, wherein said polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO:
 2. 